Biology subjects

Quake, S. R.

Publications and source records attributed to Quake, S. R..

At least 73 records · Page 4Linked to original sources

bioRxiv2020

Single-cell analysis of Schistosoma mansoni reveals a conserved genetic program controlling germline stem cell fate

Schistosomes are parasitic flatworms causing one of the most prevalent infectious diseases from which millions of people are currently suffering. Their germline outputs many fertilized eggs, which are both the transmissible agents and the cause of the infection-associated pathology. Given its significance, the schistosome germline has been a research focus for more than a century. Nonetheless, little is known about the molecular mechanisms that regulate its development. Here, we construct a transcriptomic cell type atlas of juvenile schistosomes. This allows us to capture germline stem cells (GSCs) during de novo gonadal development. We identify a genetic program that balances the fate of GSC between proliferation and differentiation. This program is controlled by onecut, a homeobox transcription factor, and boule, an mRNA binding protein. Evaluating this genetic program in schistosomes free-living evolutionary cousin, the planarian, shows that this germline-specific regulatory program is conserved but its function has changed significantly during evolution.

developmental biology↗

bioRxiv2020

Single-cell transcriptional atlas of the Chinese horseshoe bat (Rhinolophus sinicus) provides insight into the cellular mechanisms which enable bats to be viral reservoirs

Bats are a major "viral reservoir" in nature and there is a great interest in not only the cell biology of their innate and adaptive immune systems, but also in the expression patterns of receptors used for cellular entry by viruses with potential cross-species transmission. To address this and other questions, we created a single-cell transcriptomic atlas of the Chinese horseshoe bat (Rhinolophus sinicus) which comprises 82,924 cells from 19 organs and tissues. This atlas provides a molecular characterization of numerous cell types from a variety of anatomical sites, and we used it to identify clusters of transcription features that define cell types across all of the surveyed organs. Analysis of viral entry receptor genes for known zoonotic viruses showed cell distribution patterns similar to that of humans, with higher expression levels in bat intestine epithelial cells. In terms of the immune system, CD8+ T cells are in high proportion with tissue-resident memory T cells, and long-lived effector memory nature killer (NK) T-like cells (KLRG1, GZMA and ITGA4 genes) are broadly distributed across the organs. Isolated lung primary bat pulmonary fibroblast (BPF) cells were used to evaluate innate immunity, and they showed a weak response to interferon {beta} and tumor necrosis factor- compared to their human counterparts, consistent with our transcriptional analysis. This compendium of transcriptome data provides a molecular foundation for understanding the cell identities, functions and cellular receptor characteristics for viral reservoirs and zoonotic transmission.

cell biology↗

bioRxiv2020

Cerebellar nuclei evolved by repeatedly duplicating a conserved cell type set

How have complex brains evolved from simple circuits? Here we investigated brain region evolution at cell type resolution in the cerebellar nuclei (CN), the output structures of the cerebellum. Using single-nucleus RNA sequencing in mice, chickens, and humans, as well as STARmap spatial transcriptomic analysis and whole-CNS projection tracing in mice, we identified a conserved cell type set containing two classes of region-specific excitatory neurons and three classes of region-invariant inhibitory neurons. This set constitutes an archetypal CN that was repeatedly duplicated to form new regions. Interestingly, the excitatory cell class that preferentially funnels information to lateral frontal cortices in mice becomes predominant in the massively expanded human Lateral CN. Our data provide the first characterization of CN transcriptomic cell types in three species and suggest a model of brain region evolution by duplication and divergence of entire cell type sets.

neuroscience↗

bioRxiv2020

Latrophilin-2 repels Teneurin-3+ hippocampal axons during target selection

Parallel information processing is a salient feature of complex nervous systems. For example, the medial and lateral hippocampal networks (MHN and LHN) preferentially process spatial- and object-related information, respectively. However, the mechanisms underlying parallel network assembly during development remain largely unknown. Here, we show that complementary expression of cell-surface molecules Teneurin-3 (Ten3) and Latrophilin-2 (Lphn2) in the MHN and LHN, respectively, guides the precise assembly of both the MHN and LHN. Viral-genetic perturbations in vivo demonstrate that Ten3+ axons are repelled by target-derived Lphn2, revealing that Lphn2/Ten3-mediated repulsion and Ten3/Ten3-mediated attraction cooperate to control precise target selection of MHN axons. In the LHN, Lphn2+ axons are confined to Lphn2+ targets via repulsion from Ten3+ targets. Our findings demonstrate that assembly of parallel hippocampal networks follows a Ten3[->]Ten3, Lphn2[->]Lphn2 rule instructed by reciprocal repulsions.

neuroscience↗

bioRxiv2020

Differential encoding in prefrontal cortex projection neuron classes across cognitive tasks

Single-cell transcriptomics has been widely applied to classify neurons in the mammalian brain, while systems neuroscience has historically analyzed the encoding properties of cortical neurons without considering cell types. Here we examine how specific transcriptomic types of mouse prefrontal cortex (PFC) projection neurons relate to axonal projections and encoding properties across multiple cognitive tasks. We found that most types projected to multiple targets, and most targets received projections from multiple types, except PFC[->]PAG (periaqueductal gray). By comparing Ca2+-activity of the molecularly homogeneous PFC[->]PAG type against two heterogeneous classes in several two-alternative choice tasks in freely-moving mice, we found that all task-related signals assayed were qualitatively present in all examined classes. However, PAG-projecting neurons most potently encoded choice in cued tasks, whereas contralateral PFC-projecting neurons most potently encoded reward context in an uncued task. Thus, task signals are organized redundantly, but with clear quantitative biases across cells of specific molecular-anatomical characteristics.

neuroscience↗

bioRxiv2020

The transcriptional landscape of Venezuelan equine encephalitis virus infection

Venezuelan Equine Encephalitis Virus (VEEV) is a major biothreat agent that naturally causes outbreaks in humans and horses particularly in tropical areas of the western hemisphere, for which no antiviral therapy is currently available. The host response to VEEV and the cellular factors this alphavirus hijacks to support its effective replication or evade cellular immune responses are largely uncharacterized. We have previously demonstrated tremendous cell-to-cell heterogeneity in viral RNA (vRNA) and cellular transcript levels during flaviviral infection using a novel virus-inclusive single-cell RNA-Seq approach. Here, we used this unbiased, genome-wide approach to simultaneously profile the host transcriptome and vRNA in thousands of single cells during infection of human astrocytes with the live-attenuated vaccine strain of VEEV (TC-83). Host transcription was profoundly suppressed, yet "superproducer cells" with extremely high vRNA abundance emerged during the first viral life cycle and demonstrated an altered transcriptome relative to both uninfected cells and cells with high vRNA abundance harvested at later time points. Additionally, cells with increased structural-to-nonstructural transcript ratio exhibited upregulation of intracellular membrane trafficking genes at later time points. Loss- and gain-of-function experiments confirmed pro- and antiviral activities in both vaccine and virulent VEEV infections among the products of transcripts that positively or negatively correlated with vRNA abundance, respectively. Lastly, comparison with single cell transcriptomic data from other viruses highlighted common and unique pathways perturbed by infection across evolutionary scales. This study provides a high-resolution characterization of the VEEV (TC-83)-host interplay, identifies candidate targets for antivirals, and establishes a comparative single-cell approach to study the evolution of virus-host interactions. Author SummaryLittle is known about the host response to Venezuelan Equine Encephalitis Virus (VEEV) and the cellular factors this alphavirus hijacks to support effective replication or evade cellular immune responses. Monitoring dynamics of host and viral RNA (vRNA) during viral infection at a single-cell level can provide insight into the virus-host interplay at a high resolution. Here, a single-cell RNA sequencing technology that detects host and viral RNA was used to investigate the interactions between TC-83, the vaccine strain of VEEV, with the human host during the course of infection of U-87 MG cells (human astrocytoma). Virus abundance and host transcriptome were heterogeneous across cells from the same culture. Subsets of differentially expressed genes, positively or negatively correlating with vRNA abundance, were identified and subsequently in vitro validated as candidate proviral and antiviral factors, respectively, in TC-83 and/or virulent VEEV infections. In the first replication cycle, "superproducer" cells exhibited rapid increase in vRNA abundance and unique gene expression patterns. At later time points, cells with increased structural-to-nonstructural transcript ratio demonstrated upregulation of intracellular membrane trafficking genes. Lastly, comparing the VEEV dataset with published datasets on other RNA viruses revealed unique and overlapping responses across viral clades. Overall, this study improves the understanding of VEEV-host interactions, reveals candidate targets for antiviral approaches, and establishes a comparative single-cell approach to study the evolution of virus-host interactions.

microbiology↗

bioRxiv2020

Form and function: diverse homeostatic and immunomodulatory roles of immune cells in the developing lung revealed at single cell resolution

At birth, the lungs experience a sudden transition from a pathogen-free, hypoxic, fluid-filled environment to a pathogen-rich, rhythmically distended air-liquid interface. While many studies focus on adult tissue, the heterogeneity of immune cells in the perinatal lung remains unexplored. Here, we combine single cell transcriptomics with in situ hybridization to present an atlas of the murine lung immune compartment during a critical period of lung development. We show that the late embryonic lung is dominated by specialized proliferative macrophages with a surprising physical interaction with the developing vasculature. These macrophages disappear after birth and are replaced by a complex and dynamic mixture of macrophage subtypes, dendritic cells, granulocytes, and lymphocytes. Detailed characterization of macrophage diversity revealed a precise orchestration of five distinct subpopulations across postnatal development to fill context-specific functions in tissue remodeling, angiogenesis, and immunity. These data both broaden the putative roles for immune cells in the developing lung and provide a framework for understanding how external insults alter immune cell phenotype during a period of rapid lung growth and heightened vulnerability.

developmental biology↗

bioRxiv2020

Differential Impacts on Host Transcription by ROP and GRA Effectors from the Intracellular Parasite Toxoplasma gondii

The intracellular parasite Toxoplasma gondii employs a vast array of effector proteins from the rhoptry and dense granule organelles to modulate host cell biology; these effectors are known as ROPs and GRAs, respectively. To examine the individual impacts of ROPs and GRAs on host gene expression, we developed a robust, novel protocol to enrich for ultra-pure populations of a naturally occurring and reproducible population of host cells called uninfected-injected (U-I) cells, which Toxoplasma injects with ROPs but subsequently fails to invade. We then performed single cell transcriptomic analysis at 1-3 hours post-infection on U-I cells (as well as on uninfected and infected controls) arising from infection with either wild type parasites or parasites lacking the MYR1 protein, which is required for soluble GRAs to cross the parasitophorous vacuole membrane (PVM) and reach the host cell cytosol. Based on comparisons of infected and U-I cells, the hosts earliest response to infection appears to be driven primarily by the injected ROPs, which appear to induce immune and cellular stress pathways. These ROP-dependent pro-inflammatory signatures appear to be counteracted by at least some of the MYR1-dependent GRAs and may be enhanced by the MYR-independent GRAs, (which are found embedded within the PVM). Finally, signatures detected in uninfected bystander cells from the infected monolayers suggests that MYR1-dependent paracrine effects also counteract inflammatory ROP-dependent processes. IMPORTANCEThis work performs the first transcriptomic analysis of U-I cells, captures the earliest stage of a host cells interaction with Toxoplasma gondii, and dissects the effects of individual classes of parasite effectors on host cell biology.

microbiology↗

bioRxiv2019

NFIA regulates granule recruitment and exocytosis in the adult pancreas

After food ingestion, pancreatic cells secrete zymogen and hormone-containing granules to precisely control digestion and blood glucose levels. Identifying regulators of this process is paramount to combatting multiple pancreatic diseases. Here we show that pancreatic deletion of the transcription factor nuclear factor IA (NFIA) leads to hyperglycemia, hypoinsulinemia, and hypolipidemia. Surprisingly, insulin and digestive enzymes are produced in the absence of NFIA, however, they are not secreted properly and instead accumulate inside pancreatic cells. In NFIA-deficient mice we saw a reduction of insulin granules in the ready releasable pool and the first-phase insulin response was impaired. We found that NFIA binds to and activates Rab39b, a Rab GTPase critical for exocytosis. Re-expression of Rab39b in NFIA knockout islets restored glucose-stimulated insulin secretion. In sum, the NFIA-Rab39b axis regulates pancreatic physiology through granule recruitment and docking, linking NFIA to a new process with potential effects in diabetes, pancreatitis, and lipid disorders.

cell biology↗

bioRxiv2019

northstar: leveraging cell atlases to identify healthy and neoplastic cells in transcriptomes from human tumors

Single cell transcriptomics is revolutionising our understanding of tissue and disease heterogeneity, yet cell type identificationl remains a partially manual task. Published algorithms for automatic cell annotation are limited to known cell types and fail to capture novel populations, especially cancer cells. We developed northstar, a computational approach to classify thousands of cells based on published data within seconds while simultaneously identifying and highlighting new cell states such as malignancies. We tested northstar on human glioblastoma and melanoma and obtained high accuracy and robustness. We collected eleven pancreatic tumors and identified three shared and five private neoplastic cell populations, offering insight into the origins of neuroendocrine and exocrine tumors. northstar is a useful tool to assign known and novel cell type and states in the age of cell atlases.

bioinformatics↗

bioRxiv2019

Cell-Surface Proteomic Profiling in the Fly Brain Uncovers New Wiring Regulators

Molecular interactions at the cellular interface mediate organized assembly of single cells into tissues, and thus govern the development and physiology of multicellular organisms. Here, we developed a cell-type-specific, spatiotemporally-resolved approach to profile cell-surface proteomes in intact tissues. Quantitative profiling of cell-surface proteomes of Drosophila olfactory projection neurons (PNs) in pupae and adults revealed a global down-regulation of wiring molecules and an up-regulation of synaptic molecules in the transition from developing to mature PNs. A proteome-instructed in vivo screen identified 20 new cell-surface molecules regulating neural circuit assembly, many of which belong to evolutionarily conserved protein families not previously linked to neural development. Genetic analysis further revealed that the lipoprotein receptor LRP1 cell-autonomously controls PN dendrite targeting, contributing to the formation of a precise olfactory map. These findings highlight the power of temporally-resolved in situ cell-surface proteomic profiling in discovering new regulators of brain wiring.

neuroscience↗

bioRxiv2019

Molecular and Morphological Signatures of Chordate Development: Two Distinct Pathways, One Tunicate

All chordates, including urochordates such as tunicates, develop through embryogenesis. The chordate larvae of colonial tunicates metamorphose to lose all chordate structures such as notochord, neural tube, segmented musculature, and then develop by asexual reproduction [blastogenesis], whereby stem cells form tissues and organs. These two developmental pathways establish the same body axis, morphogenetic patterning and organ formation. It is unknown if this convergent morphology implies convergent cellular and molecular mechanisms, and whether the stem cells that mediate these processes differ. Using the colonial tunicate Botryllus schlosseri, we combined transcriptome sequencing and multiple microscopy techniques to study the molecular and morphological signatures of cells at each developmental stage of embryogenesis and blastogenesis. This revealed that the molecular programs are distinct, but the blastogenic tissue-specific stem cells and embryonic precursor populations share similar molecular profiles. By comparing embryogenesis in other chordates we found shared developmental principles, highlighting transcription factors as key evolutionary conserved elements. This study establishes a platform for advancing the science of stem cell biology and regulation of development and regeneration.

developmental biology↗

bioRxiv2019

Functional characterization and lineage analysis of broadly neutralizing human antibodies against dengue virus identified by single B cell transcriptomics

Eliciting broadly neutralizing antibodies (bNAbs) against the four dengue virus serotypes (DENV1-4) that are spreading into new territories is an important goal of vaccine design. To delineate bNAb targets, we characterized 28 monoclonal antibodies belonging to expanded and hypermutated clonal families identified by transcriptomic analysis of single plasmablasts from DENV-infected individuals. Among these, we identified two somatically related bNAbs that potently neutralized DENV1-4. Mutagenesis studies revealed that the major recognition determinants of these bNAbs are in E protein domain I, distinct from the only known class of human bNAbs against flaviviruses with a well-defined epitope. B cell repertoire analysis from acute-phase peripheral blood suggested a memory origin and divergent somatic hypermutation pathways for these bNAbs, and a limited number of mutations was sufficient for neutralizing activity. Our study suggests multiple B cell evolutionary pathways leading to DENV bNAbs targeting a novel epitope that can be exploited for vaccine design.

microbiology↗

bioRxiv2019

A T cell behavioral manifold couples speed and turning to create heterogeneity in vivo

T cells in vivo migrate primarily via undirected random walks, but it remains unresolved how these random walks generate an efficient search. Here, we use light sheet microscopy of T cells in the larval zebrafish as a model system to study motility across large populations of cells over hours in their native context. We show that cell-to-cell variability is amplified by a correlation between speed and directional persistence, generating a characteristic cell behavioral manifold that is preserved under a perturbation to cell speeds, and seen in Mouse T cells and Dictyostelium. These results suggest that there is a single variable underlying ameboid cell motility that jointly controls speed and turning. This coupling explains behavioral heterogeneity in diverse systems and allows cells to access a broad range of length scales.

biophysics↗

bioRxiv2019

Persistent transcriptional programs are associated with remote memory in diverse cells of the medial prefrontal cortex

It is thought that memory is stored in engrams, a subset of neurons that undergo learning-induced alterations. The role of gene-expression during learning and short-term memory has been studied extensively, but little is known about remote memory that can persist for a lifetime. Using long-term contextual fear memory as a paradigm, an activity-dependent transgenic model for engram-specific labeling, and single-cell transcriptomics we probed the gene-expression landscape underlying remote memory consolidation and recall in the medial prefrontal cortex. Remarkably, we find sustained activity-specific transcriptional alterations in diverse populations of neurons that persist even weeks after fear-learning and are distinct from those previously identified in learning. Out of a vast plasticity-coding space, we uncover select membrane-fusion genes that could play important roles in maintaining remote memory traces. Unexpectedly, astrocytes and microglia also acquire new persistent gene signatures upon recall of remote memory, suggesting that they actively contribute to memory circuits. Our discovery of novel distinct gene-expression programs involved in long term memory adds an important dimension of activity-dependent cellular states to existing brain taxonomy atlases and sheds light on the elusive mechanisms of remote memory storage.

neuroscience↗

bioRxiv2019

A molecular cell atlas of the human lung from single cell RNA sequencing

Although single cell RNA sequencing studies have begun providing compendia of cell expression profiles, it has proven more difficult to systematically identify and localize all molecular cell types in individual organs to create a full molecular cell atlas. Here we describe droplet- and plate-based single cell RNA sequencing applied to [~]75,000 human lung and blood cells, combined with a multi-pronged cell annotation approach, which have allowed us to define the gene expression profiles and anatomical locations of 58 cell populations in the human lung, including 41 of 45 previously known cell types or subtypes and 14 new ones. This comprehensive molecular atlas elucidates the biochemical functions of lung cell types and the cell-selective transcription factors and optimal markers for making and monitoring them; defines the cell targets of circulating hormones and predicts local signaling interactions including sources and targets of chemokines in immune cell trafficking and expression changes on lung homing; and identifies the cell types directly affected by lung disease genes and respiratory viruses. Comparison to mouse identified 17 molecular types that appear to have been gained or lost during lung evolution and others whose expression profiles have been substantially altered, revealing extensive plasticity of cell types and cell-type-specific gene expression during organ evolution including expression switches between cell types. This atlas provides the molecular foundation for investigating how lung cell identities, functions, and interactions are achieved in development and tissue engineering and altered in disease and evolution.

genomics↗

bioRxiv2019

Molecular and genetic regulation of pig pancreatic islet cell development

Reliance on rodents for understanding pancreatic genetics, development and islet function could limit progress in developing interventions for human diseases like diabetes mellitus. Similarities of pancreas morphology and function suggest that porcine and human pancreas developmental biology may have useful homologies. However, little is known about pig pancreas development. To fill this knowledge gap, we investigated fetal and neonatal pig pancreas at multiple, crucial developmental stages using modern experimental approaches. Purification of islet {beta}-, - and {delta}-cells followed by transcriptome analysis (RNA-Seq) and immunohistology identified cell- and stage-specific regulation, and revealed that pig and human islet cells share characteristic features not observed in mice. Morphometric analysis also revealed endocrine cell allocation and architectural similarities between pig and human islets. Our analysis unveiled scores of signaling pathways linked to native islet {beta}-cell functional maturation, including evidence of fetal -cell GLP-1 production and signaling to {beta}-cells. Thus, the findings and resources detailed here show how pig pancreatic islet studies complement other systems for understanding the developmental programs that generate functional islet cells, and that are relevant to human pancreatic diseases. Summary StatementThis study reveals transcriptional, signaling and cellular programs governing pig pancreatic islet development, including striking similarities to human islet ontogeny, providing a novel resource for advancing human islet replacement strategies.

developmental biology↗

bioRxiv2019

Transcriptional program of memory B cell activation, broadly binding anti-influenza antibodies, and bystander activation after vaccination revealed by single-cell transcriptomics

Antibody memory protects humans from many diseases. Protective antibody memory responses require activation of transcriptional programs, cell proliferation, and production of antigen-specific antibodies, but how these aspects of the response are coordinated is poorly understood. We profiled the molecular and cellular features of the antibody response to influenza vaccination by integrating single-cell transcriptomics, longitudinal antibody repertoire sequencing, and antibody binding measurements. Single-cell transcriptional profiling revealed a program of memory B cell activation characterized by CD11c and T-bet expression associated with clonal expansion and differentiation toward effector function. Vaccination elicited an antibody clone which rapidly acquired broad high-affinity hemagglutinin binding during affinity maturation. Unexpectedly, many antibody clones elicited by vaccination do not bind vaccine, demonstrating non-specific activation of bystander antibodies by influenza vaccination. These results offer insight into how molecular recognition, transcriptional programs, and clonal proliferation are coordinated in the human B cell repertoire during memory recall.

immunology↗